U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

How cancer doctors use personalized medicine to target variations unique to each tumour

The Children’s Cancer Institute in Sydney recently launched an ambitious program. From early next year, scientists will analyse the unique cancer cells of 12 children diagnosed with the most aggressive forms of the disease to find the best treatment for each child. By 2020, they aim to have these individualised treatment options available to all children diagnosed with cancers that have a less than 30% survival rate. This way of tailoring treatment to each person is known as personalised medicine, and advances in DNA sequencing have paved the way for a new era in cancer management.

Evaluation of polymeric nanomedicines targeted to PSMA: Effect of ligand on targeting efficiency

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA−). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Development of an efficiency equation for solar evaporator- collectors

Considering the growing energy needs and concern for environmental degradation, clean and inexhaustible energy sources, e.g solar energy are receiving greater attention for various applications. The use of solar energy systems for low temperature applications reduces the burden on conventional fossil fuels and has little or no harmful effects on the environment. The performance of a solar system depends to a great extent on the collector used for the conversion of solar radiant energy to thermal energy. A solar evaporatorcollector (SEC) is basically an unglazed flat plate collector where refrigerant, like R134a, is used as the working fluid. As the operating temperature of SEC is very low, it collects energy both from solar irradiation and ambient energy leading to a much higher efficiency than the conventional collectors. The capability of SEC to utilize ambient energy also enables the system to operate at night. Therefore it is not appropriate to use for the evaluation of performance of SEC by conventional efficiency equation where ambient energy and condensation is not considered as energy input in addition to irradiation. In the National University of Singapore, several Solar Assisted Heat Pump (SAHP) systems were built for the evaluation of performance under the metrological condition of Singapore for thermal applications of desalination and SEC was the main component to harness renewable energy. In this paper, the design and performance of SEC are explored. Furthermore, an attempt is made to develop an efficiency equation for SEC and maximum efficiency attained 98% under the meteorological condition of Singapore.

Low-impact Use: A New Standard

Once the ugly duckling of the lighting world, the fluorescent bulb recently has become something of an eco-darling thanks to its energy efficiency. Whereas a standard off-the-shelf incandescent bulb devotes only about five percent of its total electrical consumption to produce visible light (the remainder is released in heat), fluorescent lighting employs an entirely different process (it radiates rather than burns) that is four to six times more efficient. Fluorescents are indisputably superior in ­performance, but up to 5 milligrams of mercury, a hazardous trace metal, is included in the manufacture of each lamp

Straw Performance Studies and Quality Assurance for the ATLAS Transition Radiation Tracker

The Transition Radiation Tracker (TRT) of the ATLAS experiment at the LHC is part of the Inner Detector. It is designed as a robust and powerful gaseous detector that provides tracking through individual drift-tubes (straws) as well as particle identification via transition radiation (TR) detection. The straw tubes are operated with Xe-CO2-O2 70/27/3, a gas that combines the advantages of efficient TR absorption, a short electron drift time and minimum ageing effects. The modules of the barrel part of the TRT were built in the United States while the end-cap wheels are assembled at two Russian institutes. Acceptance tests of barrel modules and end-cap wheels are performed at CERN before assembly and integration with the Semiconductor Tracker (SCT) and the Pixel Detector. This thesis first describes simulations the TRT straw tube. The argon-based acceptance gas mixture as well as two xenon-based operating gases are examined for its properties. Drift velocities and Townsend coefficients are computed with the help of the program Magboltz and used to study electron drift and multiplication in the straw using the software Garfield. The inclusion of Penning transfers in the avalanche process leads to remarkable agreements with experimental data. A high level of cleanliness in the TRT s acceptance test gas system is indispensable. To monitor gas purity, a small straw tube detector has been constructed and extensively used to study the ageing behaviour of the straw tube in Ar-CO2. A variety of ageing tests are presented and discussed. Acceptance tests for the TRT survey dimensions, wire tension, gas-tightness, high-voltage stability and gas gain uniformity along each individual straw. The thesis gives details on acceptance criteria and measurement methods in the case of the end-cap wheels. Special focus is put on wire tension and straw straightness. The effect of geometrically deformed straws on gas gain and energy resolution is examined in an experimental setup and compared to simulation studies. An overview of the most important results from the end-cap wheels tested up to this point is presented.

Patient exposure monitoring and radiation qualities in two-dimensional digital x-ray imaging

The methods for estimating patient exposure in x-ray imaging are based on the measurement of radiation incident on the patient. In digital imaging, the useful dose range of the detector is large and excessive doses may remain undetected. Therefore, real-time monitoring of radiation exposure is important. According to international recommendations, the measurement uncertainty should be lower than 7% (confidence level 95%). The kerma-area product (KAP) is a measurement quantity used for monitoring patient exposure to radiation. A field KAP meter is typically attached to an x-ray device, and it is important to recognize the effect of this measurement geometry on the response of the meter. In a tandem calibration method, introduced in this study, a field KAP meter is used in its clinical position and calibration is performed with a reference KAP meter. This method provides a practical way to calibrate field KAP meters. However, the reference KAP meters require comprehensive calibration. In the calibration laboratory it is recommended to use standard radiation qualities. These qualities do not entirely correspond to the large range of clinical radiation qualities. In this work, the energy dependence of the response of different KAP meter types was examined. According to our findings, the recommended accuracy in KAP measurements is difficult to achieve with conventional KAP meters because of their strong energy dependence. The energy dependence of the response of a novel large KAP meter was found out to be much lower than with a conventional KAP meter. The accuracy of the tandem method can be improved by using this meter type as a reference meter. A KAP meter cannot be used to determine the radiation exposure of patients in mammography, in which part of the radiation beam is always aimed directly at the detector without attenuation produced by the tissue. This work assessed whether pixel values from this detector area could be used to monitor the radiation beam incident on the patient. The results were congruent with the tube output calculation, which is the method generally used for this purpose. The recommended accuracy can be achieved with the studied method. New optimization of radiation qualities and dose level is needed when other detector types are introduced. In this work, the optimal selections were examined with one direct digital detector type. For this device, the use of radiation qualities with higher energies was recommended and appropriate image quality was achieved by increasing the low dose level of the system.

Reconstruction of past UV radiation

Solar ultraviolet (UV) radiation has a broad range of effects concerning life on Earth. Soon after the mid-1980s, it was recognized that the stratospheric ozone content was declining over large areas of the globe. Because the stratospheric ozone layer protects life on Earth from harmful UV radiation, this lead to concern about possible changes in the UV radiation due to anthropogenic activity. Initiated by this concern, many stations for monitoring of the surface UV radiation were founded in the late 1980s and early 1990s. As a consequence, there is an apparent lack of information on UV radiation further in the past: measurements cannot tell us how the UV radiation levels have changed on time scales of, for instance, several decades. The aim of this thesis was to improve our understanding of past variations in the surface UV radiation by developing techniques for UV reconstruction. Such techniques utilize commonly available meteorological data together with measurements of the total ozone column for reconstructing, or estimating, the amount of UV radiation reaching Earth's surface in the past. Two different techniques for UV reconstruction were developed. Both are based on first calculating the clear-sky UV radiation using a radiative transfer model. The clear-sky value is then corrected for the effect of clouds based on either (i) sunshine duration or (ii) pyranometer measurements. Both techniques account also for the variations in the surface albedo caused by snow, whereas aerosols are included as a typical climatological aerosol load. Using these methods, long time series of reconstructed UV radiation were produced for five European locations, namely Sodankylä and Jokioinen in Finland, Bergen in Norway, Norrköping in Sweden, and Davos in Switzerland. Both UV reconstruction techniques developed in this thesis account for the greater part of the factors affecting the amount of UV radiation reaching the Earth's surface. Thus, they are considered reliable and trustworthy, as suggested also by the good performance of the methods. The pyranometer-based method shows better performance than the sunshine-based method, especially for daily values. For monthly values, the difference between the performances of the methods is smaller, indicating that the sunshine-based method is roughly as good as the pyranometer-based for assessing long-term changes in the surface UV radiation. The time series of reconstructed UV radiation produced in this thesis provide new insight into the past UV radiation climate and how the UV radiation has varied throughout the years. Especially the sunshine-based UV time series, extending back to 1926 and 1950 at Davos and Sodankylä, respectively, also put the recent changes driven by the ozone decline observed over the last few decades into perspective. At Davos, the reconstructed UV over the period 1926-2003 shows considerable variation throughout the entire period, with high values in the mid-1940s, early 1960s, and in the 1990s. Moreover, the variations prior to 1980 were found to be caused primarily by variations in the cloudiness, while the increase of 4.5 %/decade over the period 1979-1999 was supported by both the decline in the total ozone column and changes in the cloudiness. Of the other stations included in this work, both Sodankylä and Norrköping show a clear increase in the UV radiation since the early 1980s (3-4 %/decade), driven primarily by changes in the cloudiness, and to a lesser extent by the diminution of the total ozone. At Jokioinen, a weak increase was found, while at Bergen there was no considerable overall change in the UV radiation level.

Characterisation of properties of coniferous wood tracheids by x-ray diffraction, laser scattering and microscopy

In recent years there has been growing interest in selecting suitable wood raw material to increase end product quality and to increase the efficiency of industrial processes. Genetic background and growing conditions are known to affect properties of growing trees, but only a few parameters reflecting wood quality, such as volume and density can be measured on an industrial scale. Therefore research on cellular level structures of trees grown in different conditions is needed to increase understanding of the growth process of trees leading to desired wood properties. In this work the cellular and cell wall structures of wood were studied. Parameters, such as the mean microfibril angle (MFA), the spiral grain angles, the fibre length, the tracheid cell wall thickness and the cross-sectional shape of the tracheid, were determined as a function of distance from the pith towards the bark and mutual dependencies of these parameters were discussed. Samples from fast-grown trees, which belong to a same clone, grown in fertile soil and also from fertilised trees were measured. It was found that in fast-grown trees the mean MFA decreased more gradually from the pith to the bark than in reference stems. In fast-grown samples cells were shorter, more thin-walled and their cross-sections were rounder than in slower-grown reference trees. Increased growth rate was found to cause an increase in spiral grain variation both within and between annual rings. Furthermore, methods for determination of the mean MFA using x-ray diffraction were evaluated. Several experimental arrangements including the synchrotron radiation based microdiffraction were compared. For evaluation of the data analysis procedures a general form for diffraction conditions in terms of angles describing the fibre orientation and the shape of the cell was derived. The effects of these parameters on the obtained microfibril angles were discussed. The use of symmetrical transmission geometry and tangentially cut samples gave the most reliable MFA values.

Lung structure and function studied by synchrotron radiation

A novel method for functional lung imaging was introduced by adapting the K-edge subtraction method (KES) to in vivo studies of small animals. In this method two synchrotron radiation energies, which bracket the K-edge of the contrast agent, are used for simultaneous recording of absorption-contrast images. Stable xenon gas is used as the contrast agent, and imaging is performed in projection or computed tomography (CT) mode. Subtraction of the two images yields the distribution of xenon, while removing practically all features due to other structures, and the xenon density can be calculated quantitatively. Because the images are recorded simultaneously, there are no movement artifacts in the subtraction image. Time resolution for a series of CT images is one image/s, which allows functional studies. Voxel size is 0.1mm3, which is an order better than in traditional lung imaging methods. KES imaging technique was used in studies of ventilation distribution and the effects of histamine-induced airway narrowing in healthy, mechanically ventilated, and anaesthetized rabbits. First, the effect of tidal volume on ventilation was studied, and the results show that an increase in tidal volume without an increase in minute ventilation results a proportional increase in regional ventilation. Second, spiral CT was used to quantify the airspace volumes in lungs in normal conditions and after histamine aerosol inhalation, and the results showed large patchy filling defects in peripheral lungs following histamine provocation. Third, the kinetics of proximal and distal airway response to histamine aerosol were examined, and the findings show that the distal airways react immediately to histamine and start to recover, while the reaction and the recovery in proximal airways is slower. Fourth, the fractal dimensions of lungs was studied, and it was found that the fractal dimension is higher at the apical part of the lungs compared to the basal part, indicating structural differences between apical and basal lung level. These results provide new insights to lung function and the effects of drug challenge studies. Nowadays the technique is available at synchrotron radiation facilities, but the compact synchrotron radiation sources are being developed, and in relatively near future the method may be used at hospitals.

Modeling of Surface UV Radiation Using Satellite Data

Solar UV radiation is harmful for life on planet Earth, but fortunately the atmospheric oxygen and ozone absorb almost entirely the most energetic UVC radiation photons. However, part of the UVB radiation and much of the UVA radiation reaches the surface of the Earth, and affect human health, environment, materials and drive atmospheric and aquatic photochemical processes. In order to quantify these effects and processes there is a need for ground-based UV measurements and radiative transfer modeling to estimate the amounts of UV radiation reaching the biosphere. Satellite measurements with their near-global spatial coverage and long-term data conti-nuity offer an attractive option for estimation of the surface UV radiation. This work focuses on radiative transfer theory based methods used for estimation of the UV radiation reaching the surface of the Earth. The objectives of the thesis were to implement the surface UV algorithm originally developed at NASA Goddard Space Flight Center for estimation of the surface UV irradiance from the meas-urements of the Dutch-Finnish built Ozone Monitoring Instrument (OMI), to improve the original surface UV algorithm especially in relation with snow cover, to validate the OMI-derived daily surface UV doses against ground-based measurements, and to demonstrate how the satellite-derived surface UV data can be used to study the effects of the UV radiation. The thesis consists of seven original papers and a summary. The summary includes an introduction of the OMI instrument, a review of the methods used for modeling of the surface UV using satellite data as well as the con-clusions of the main results of the original papers. The first two papers describe the algorithm used for estimation of the surface UV amounts from the OMI measurements as well as the unique Very Fast Delivery processing system developed for processing of the OMI data received at the Sodankylä satellite data centre. The third and the fourth papers present algorithm improvements related to the surface UV albedo of the snow-covered land. Fifth paper presents the results of the comparison of the OMI-derived daily erythemal doses with those calculated from the ground-based measurement data. It gives an estimate of the expected accuracy of the OMI-derived sur-face UV doses for various atmospheric and other conditions, and discusses the causes of the differences between the satellite-derived and ground-based data. The last two papers demonstrate the use of the satellite-derived sur-face UV data. Sixth paper presents an assessment of the photochemical decomposition rates in aquatic environment. Seventh paper presents use of satellite-derived daily surface UV doses for planning of the outdoor material weathering tests.

Radiation effects in supported nanoparticles

Nanotechnology applications are entering the market in increasing numbers, nanoparticles being among the main classes of materials used. Particles can be used, e.g., for catalysing chemical reactions, such as is done in car exhaust catalysts today. They can also modify the optical and electronic properties of materials or be used as building blocks for thin film coatings on a variety of surfaces. To develop materials for specific applications, an intricate control of the particle properties, structure, size and shape is required. All these depend on a multitude of factors from methods of synthesis and deposition to post-processing. This thesis addresses the control of nanoparticle structure by low-energy cluster beam deposition and post-synthesis ion irradiation. Cluster deposition in high vacuum offers a method for obtaining precisely controlled cluster-assembled materials with minimal contamination. Due to the clusters small size, however, the cluster-surface interaction may drastically change the cluster properties on deposition. In this thesis, the deposition process of metal and alloy clusters on metallic surfaces is modelled using molecular dynamics simulations, and the mechanisms influencing cluster structure are identified. Two mechanisms, mechanical melting upon deposition and thermally activated dislocation motion, are shown to determine whether a deposited cluster will align epitaxially with its support. The semiconductor industry has used ion irradiation as a tool to modify material properties for decades. Irradiation can be used for doping, patterning surfaces, and inducing chemical ordering in alloys, just to give a few examples. The irradiation response of nanoparticles has, however, remained an almost uncharted territory. Although irradiation effects in nanoparticles embedded inside solid matrices have been studied, almost no work has been done on supported particles. In this thesis, the response of supported nanoparticles is studied systematically for heavy and light ion irradiation. The processes leading to damage production are identified and models are developed for both types of irradiation. In recent experiments, helium irradiation has been shown to induce a phase transformation from multiply twinned to single-crystalline nanoparticles in bimetallic alloys, but the nature of the transition has remained unknown. The alloys for which the effect has been observed are CuAu and FePt. It is shown in this thesis that transient amorphization leads to the observed transition and that while CuAu and FePt do not amorphize upon irradiation in bulk or as thin films, they readily do so as nanoparticles. This is the first time such an effect is demonstrated with supported particles, not embedded in a matrix where mixing is always an issue. An understanding of the above physical processes is essential, if nanoparticles are to be used in applications in an optimal way. This thesis clarifies the mechanisms which control particle morphology, and paves way for the synthesis of nanostructured materials tailored for specific applications.